Hepatitis C virus (HCV) is a major public healthcare burden with 3-4 million new infections occurring each year and more than 150 million individuals estimated to be chronically infected worldwide. Many of these individuals develop serious chronic liver diseases such as cirrhosis and hepatocellular carcinoma, making HCV the most frequent cause of liver transplantation.
HCV is an enveloped, positive-stranded RNA virus of the genus Hepacivirus within the Flaviviridae family. Due to a high degree of genetic heterogeneity, HCV has been classified in 6 epidemiologically important genotypes and numerous subtypes, differing in approximately 30% and 20% of their nucleotide and amino acid sequence, respectively.
Genotypes show important clinical and biological differences. Serotypes have not been defined; however, different genotypes and subtypes show differential sensitivity to neutralizing antibodies found in sera of chronically infected patients and to monoclonal neutralizing antibodies with therapeutic potential.
The 9.6 kb HCV genome consists of 5′ and 3′ untranslated regions and a single open reading frame encoding structural proteins (Core, E1 and E2), the viroporin p7, and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). The HCV virion is believed to consist of a nucleocapsid of HCV Core proteins containing the genomic RNA, covered by a lipid envelope with the HCV envelope glycoproteins E1 and E2. The HCV life cycle is tightly linked to the hepatic lipid metabolism.
During assembly and release, the HCV virion is believed to associate with very-low-density-lipoprotein (VLDL) or VLDL-like structures, creating lipo-viro-particles (LVP). Thus, HCV apparently circulates in infected patients associated to different classes of lipoproteins, resulting in a heterogeneous density profile apparent following buoyant density gradient ultracentrifugation. Components of the VLDL assembly and secretion pathway, such as apolipoprotein E (ApoE), might be important for the association between HCV and lipoproteins.
HCV entry is mediated by several co-receptors, including CD81, the low-density-lipoprotein receptor (LDLr) and the scavenger receptor class B type I (SR-BI). While HCV is believed to interact directly with CD81 through E2, interactions with other receptors, such as LDLr and SR-BI, might occur through lipoprotein components present on the LVP, such as ApoE, although direct interactions between E2 and SR-BI have also been reported. Eventually, HCV is internalized through clathrin-mediated endocytosis.
There is no vaccine available for HCV. Current standard-of-care, based on pegylated interferon-α2 and ribavirin, has limited efficacy and is associated with severe side effects and contraindications. Even though promising new compounds for treatment of HCV are being developed and licenced, only a minority of HCV-infected individuals is expected to be diagnosed and treated, mainly due to the asymptomatic nature of infection, economic constraints and contraindications.
Thus, an HCV vaccine is needed to control HCV globally. Most successful antiviral vaccines employ inactivated or attenuated whole viral particles as vaccine antigen and depend on the induction of neutralizing antibodies. Due to a lack of HCV particle-producing cell culture systems, this approach was not feasible for HCV.
Only in 2005, the first HCV cell culture system supporting the full viral life cycle was developed, based on the genotype 2a isolate JFH1 and the human hepatoma cell line Huh7 and derived cell lines.
Subsequently, culture systems producing HCV particles (HCVcc) of the major genotypes were developed using JFH1-based recombinants expressing genotype specific Core, E1, E2, p7 and NS2. Such particles could serve as antigens in a whole-virus inactivated HCV vaccine primarily aiming at induction of neutralizing antibodies against structural proteins of the major HCV genotypes.
However, HCVcc yields from the developed cell culture systems are relatively low compared to quantities envisioned to be required for vaccine production. Further, as patient derived HCV particles, HCVcc showed a heterogeneous density profile, making density-based purification and concentration procedures difficult. Also, cell cultures are typically treated with animal-derived trypsin, and growth medium used for production of HCVcc is typically supplemented with fetal bovine serum (FBS).
Vaccine development, as well as other research applications, such as biophysical studies of HCV particle composition, require generation of purified and concentrated HCVcc stocks.
This is expected to be facilitated by reducing concentrations of non-HCV proteins such as FBS derived proteins in HCVcc producing cell cultures. Further, use of FBS and animal-derived trypsin increases the risk of contamination with adventitious microbial agents, of relevance for HCV vaccine development. Thus, development of methods for production of HCVcc under serum-free conditions is a research focus.
At the onset of this study it had been demonstrated that Huh7 cells could be cultured in serum-free medium (RPMI 1640 supplemented with Na2SeO3) without previous adaptation for an extended period of time, and that serum-free cell cultures (DMEM supplemented with Na2SeO3 and lipid rich albumin) allowed replication of HCV.
However, establishment of a robust methodology for generation of high-titer single-density serum-free HCVcc is not known and is expected to aid HCV vaccine development.
This is relevant for HCV as well as other viruses similar to HCV.
It is also very important to generate new HCV recombinants that can grow at titers that are high enough to allow application for vaccine development.
Such viruses can be purified, up-concentrated and inactivated through specific procedures. These procedures are required to develop a whole virus inactivated vaccine antigen from crude cell culture supernatant.
These procedures have not been established for HCV even though the initial HCV cell culture was developed in 2005.